Dysregulated Expression and Subcellular Localization of Base Excision Repair (BER) Pathway Enzymes in Gallbladder Cancer

Base excision repair (BER) pathway is one of the repair systems that has an impact on radiotherapy and chemotherapy for cancer patients. The molecular pathogenesis of gallbladder cancer is not known extensively. In the present study we investigated whether the expression of AP endonuclease 1 (APE1) and DNA polymerase β (DNA pol β), key enzymes of BER pathway has any clinical significance with gallbladder carcinogenesis. 41 gallbladder cancer, 27 chronic cholecystitis, and 3 normal gallbladder specimens were analyzed for the expression of APE1 and DNA polymerase β by western blotting, and subcellular localization studied by immunohistochemistry. The enzymatic activity of APE1 was also studied. The correlations with expression of the above proteins with clinical-pathological characteristics of gallbladder cancer patients were analyzed. The integrated density value ratio (relative expression) of total APE1 (37 kDa + 35 kDa variant) analyzed in the three groups of tissues, was 0.76±0.03 in normal gallbladder, 0.91±0.08 in chronic cholecystitis, and 1.12±0.05 in gallbladder cancer. APE1 was found to be up-regulated in 80% of gallbladder carcinoma samples (P = 0.01). A positive trend of APE1 expression with tumor stage and lymph node positivity was observed. The enzymatic activity of APE1 was found higher in gallbladder cancer samples in comparison with chronic cholecystitis. The integrated density value ratio of DNA polymerase β for normal gallbladder, chronic cholecystitis and gallbladder cancer tissue samples were 0.46±0.03, 0.7±0.06 and 1.33±0.1, respectively. DNA polymerase β was found to be upregulated in almost all gallbladder carcinoma samples (P =0.0001), and its expression was negatively correlated with age (P=0.02). DNA polymerase β expression showed a positive trend with tumor stage and nuclear differentiation of gallbladder cancer. It may be concluded that alteration of these BER pathway proteins may be the causal factors for carcinogenesis of gallbladder, and has targeted therapeutic potential.

allbladder cancer is a rare solid tumor with poor prognosis, and almost a lethal gastrointestinal malignancy with high prevalence in certain world populations mainly India, Chile, China, and Japan (1,2). It has a median survival rate of 6 months, and 10% overall 5-years survival (3). About 70% of patients have a history of gallstone. Although there are many other known risk factors, gallstone is the most associated risk factor for this cancer (4). Since there is a lack of noticeable sign or symptoms, mostly patients are diagnosed at an advanced stage, and therefore it becomes incurable. Thus, no chemo or radiotherapy treatment has been established successfully for its advanced stage. Detailed knowledge of the molecular basis of this cancer is therefore desirable for early diagnosis and better therapeutic strategies for advanced stage.
DNA repair system maintains genomic integrity by detecting and removing DNA damage.
Base excision repair (BER) pathway is one such pathway for the removal and repair of individual base damaged by various endogenous processes such as oxidation, alkylation, and deamination (5).
AP endonuclease 1 is a multifunctional protein which is not only responsible for repair of AP sites but also acts as a redox factor maintaining transcription factors like p53, FOS, JUN, hypoxia inducible factor 1 subunit alpha (HIF-1α), paired box 5-6 (PAX5-6), and nuclear factor kappa B (NFkB), in an active reduced state (6).
Another crucial enzyme of this pathway is DNA polymerase β which plays a role in single nucleotide gap filling in BER pathway downstream to AP endonuclease 1. DNA polymerase β can be distinguished from other DNA polymerases because of lack of associated poof reading activity (7). Probably because of errorprone features, DNA polymerase β accumulates mutations in the genome, and lead to genomic instability and cancer upon overexpression in the cell. AP endonuclease 1 was reported to be elevated in gastric (8), ovarian, gastro-oesophageal, pancreatico-biliary cancers (9), medulloblastoma (10), and hepatocellular carcinoma (11). It has been shown that the knockdown of AP endonuclease 1 in cancer cell line reduces the cellular invasion and cellular doubling number, and makes the cancer cells sensitive to radiotherapy and chemotherapy by upregulating the oxidative DNA damage via NF-kB signaling pathways (12). DNA polymerase β was also found up-regulated in gastric (13), ovarian (14), and prostate cancers (15). It was reported that silencing this gene by siRNA, makes the cancer cell sensitive to chemotherapy (16).
There is limited information in gallbladder cancer regarding DNA repair pathway status and therefore, the present study has been undertaken with an aim to investigate the expression level of AP endonuclease 1 and DNA polymerase β in gallbladder cancer and to investigate the association with clinicopathological characteristics of gallbladder cancer patients.  (17). (Sigma Aldrich, USA) for equal loading, and then developed by using the above described method.

Preparation of cell-free extracts for APE1 oligonucleotide incision assay
Cell free extracts were made from chronic cholecystitis and gallbladder cancer tissue samples.

Oligonucleotide incision assay (APE1 activity assay)
The oligonucleotide incision assay was performed to estimate the APE1 incision activity by taking 45 bp DNA substrate containing "U" at its

Statistical analysis
For relative expression analysis, first the integrated density value (IDV) was calculated for each protein, and ratio was calculated by loading the control (level of actin) for respective samples.  There was no significant correlation between the expression level of total APE1 with age and metastatic condition of gallbladder cancer (Fig 2 D, F).

Sub cellular localization of APE1
Immunohistochemistry of AP endonuclease 1 showed that all normal gallbladder (3) as well as chronic cholecystitis (10)  and in the remaining 14 cases (35%) both nuclear and cytoplasmic staining was observed (Fig 3. I).
Nucleo-cytoplasmic localization was observed in those cases which showed relatively higher protein level of APE1 in western blotting.

Incision activity of APE1
Elevation in APE1 protein level and mostly teristic of gallbladder carcinoma (Fig 5).

Sub cellular localization of DNA polymerase β
Cytoplasmic localization of DNA polymerase β was observed in the normal gallbladder (3) and

Expression of 8-OHdG
We observed that oxidative DNA damage was minimal in normal gallbladder tissue (3) and chronic cholecystitis (7), whereas in gallbladder cancer tissue samples (15), the intense nuclear staining of 8-OHdG staining was observed (Fig 7).

Discussion
Accumulating evidence has demonstrated that over expression of BER proteins leads to tumor formation, less sensitivity towards radiotherapy and chemotherapy, metastasis, and a poor prognosis. This alteration in the DNA repair genes predisposes the individual to mutagens, and leads to certain types of cancer.
In conclusion, we have shown that AP endonuclease 1 and DNA polymerase β get upregulated in the gallbladder cancer and also in chronic cholecystitis in comparison with normal gallbladder. Moreover, our study suggests that these BER proteins may be potential therapeutic targets in this cancer.